Pharmaceutical compositions comprising poh derivatives

ABSTRACT

The present invention provides for a derivative of monoterpene or sesquiterpene, such as a perillyl alcohol derivative. For example, the perillyl alcohol derivative may be a perillyl alcohol carbamate. The perillyl alcohol derivative may be perillyl alcohol conjugated with a therapeutic agent such as a chemotherapeutic agent. The present invention also provides for a method of treating a disease such as cancer, comprising the step of delivering to a patient a therapeutically effective amount of a derivative of monoterpene (or sesquiterpene). The route of administration may vary, and can include, inhalation, intranasal, oral, transdermal, intravenous, subcutaneous or intramuscular injection.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application Nos.61/377,747 (filed Aug. 27, 2010) and 61/471,402 (filed Apr. 4, 2011).

FIELD OF THE INVENTION

The present invention relates to POH derivatives. The present inventionfurther relates to methods of using POH derivatives such as POHcarbamates to treat cancer.

BACKGROUND OF THE INVENTION

Malignant gliomas, the most common form of central nervous system (CNS)cancers, is currently considered essentially incurable. Among thevarious malignant gliomas, anaplastic astrocytomas (Grade III) andglioblastoma multiforme (GBM; Grade IV) have an especially poorprognosis due to their aggressive growth and resistance to currentlyavailable therapies. The present standard of care for malignant gliomasconsists of surgery, ionizing radiation, and chemotherapy. Despiterecent advances in medicine, the past 50 years have not seen anysignificant improvement in prognosis for malignant gliomas. Wen et al.Malignant gliomas in adults. New England J. Med. 359: 492-507, 2008.Stupp et al. Radiotherapy plus concomitant and adjuvant temozolomide forglioblastoma. New England J. Med. 352: 987-996, 2005.

The poor response of tumors, including malignant gliomas, to varioustypes of chemotherapeutic agents are often due to intrinsic drugresistance. Additionally, acquired resistance of initiallywell-responding tumors and unwanted side effects are other problems thatfrequently thwart long-term treatment using chemotherapeutic agents.Hence, various analogues of chemotherapeutic agents have been preparedin an effort to overcome these problems. The analogues include noveltherapeutic agents which are hybrid molecules of at least two existingtherapeutic agents. For example, cisplatin has been conjugated withPt-(II) complexes with cytotoxic codrugs, or conjugated with bioactiveshuttle components such as porphyrins, bile acids, hormones, ormodulators that expedite the transmembrane transport or the drugaccumulation within the cell. (6-Aminomethylnicotinate)dichloridoplatinum(II) complexes esterified with terpene alcohols weretested on a panel of human tumor cell lines. The terpenyl moieties inthese complexes appeared to fulfill a transmembrane shuttle function andincreased the rate and extent of the uptake of these conjugates intovarious tumor cell lines. Schobert et al. Monoterpenes as Drug Shuttles:Cytotoxic (6-minomethylnicotinate) dichloridoplatinum(II) Complexes withPotential To Overcome Cisplatin Resistance. J. Med. Chem. 2007, 50,1288-1293.

Perillyl alcohol (POH), a naturally occurring monoterpene, has beensuggested to be an effective agent against a variety of cancers,including CNS cancer, breast cancer, pancreatic cancer, lung cancer,melanomas and colon cancer. Gould, M. Cancer chemoprevention and therapyby monoterpenes. Environ Health Perspect. 1997 June; 105 (Suppl 4):977-979. Hybrid molecules containing both perillyl alcohol and retinoidswere prepared to increase apoptosis-inducing activity. Das et al. Designand synthesis of potential new apoptosis agents: hybrid compoundscontaining perillyl alcohol and new constrained retinoids. TetrahedronLetters 2010, 51, 1462-1466.

There is still a need to prepare perillyl alcohol derivatives includingperillyl alcohol conjugated with other therapeutic agents, and use thismaterial in the treatment of cancers such as malignant gliomas, as wellas other brain disorders such as Parkinson's and Alzheimer's disease.Perillyl alcohol derivatives may be administered alone or in combinationwith other treatment methods including radiation, standard chemotherapy,and surgery. The administration can also be through various routesincluding intranasal, oral, oral-tracheal for pulmonary delivery, andtransdermal.

SUMMARY OF THE INVENTION

The present invention provides for a pharmaceutical compositioncomprising a perillyl alcohol carbamate. The perillyl alcohol carbamatemay be perillyl alcohol conjugated with a therapeutic agent, such as achemotherapeutic agent. The chemotherapeutic agents that may be used inthe present invention include a DNA alkylating agent, a topoisomeraseinhibitor, an endoplasmic reticulum stress inducing agent, a platinumcompound, an antimetabolite, an enzyme inhibitor, and a receptorantagonist. In certain embodiments, the therapeutic agents are dimethylcelocoxib (DMC), temozolomide (TMZ) or rolipram. The perillyl alcoholcarbamates may be 4-(Bis-N,N′-4-isopropenyl cyclohex-1-enylmethyloxycarbonyl[5-(2,5-dimethylphenyl)-3-trifluoromethylpyrazol-1-yl]benzenesulfonamide,4-(3-cyclopentyloxy-4-methoxy phenyl)-2-oxo-pyrrolidine-1-carboxylicacid 4-isopropenyl cyclohex-1-enylmethyl ester, and 3-methyl4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carbonyl)-carbamicacid-4-isopropenyl cyclohex-1-enylmethyl ester.

The pharmaceutical compositions of the present invention may beadministered before, during or after radiation. The pharmaceuticalcompositions may be administered before, during or after theadministration of a chemotherapeutic agent. The routes of administrationof the pharmaceutical compositions include inhalation, intranasal, oral,intravenous, subcutaneous or intramuscular administration.

The invention further provides for a method for treating a disease in amammal, comprising the step of delivering to the mammal atherapeutically effective amount of a perillyl alcohol carbamate. Themethod may further comprise the step of treating the mammal withradiation, and/or further comprise the step of delivering to the mammala chemotherapeutic agent. The diseases treated may be cancer, includinga tumor of the nervous system, such as a glioblastoma. The routes ofadministration of the perillyl alcohol carbamate include inhalation,intranasal, oral, intravenous, subcutaneous or intramuscularadministration.

The present invention also provides for a process for making a POHcarbamate, comprising the step of reacting a first reactant of perillylchloroformate with a second reactant, which may be dimethyl celocoxib(DMC), temozolomide (TMZ) or rolipram. When the second reactant isdimethyl celocoxib, the reaction may be carried out in the presence ofacetone and a catalyst of potassium carbonate. When the second reactantis rolipram, the reaction may be carried out in the presence oftetrahydrofuran and a catalyst of n-butyl lithium. The perillylchloroformate may also be prepared by reacting perillyl alcohol withphosgene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of the MTT cytotoxicity assays demonstratingthe efficacy of dimethyl celecoxib (DMC) in killing U87, A172 and U251human glioma cells.

FIG. 2 shows the results of the MTT cytotoxicity assays demonstratingthe efficacy of the POH-DMC conjugate in killing U87, A172 and U251human glioma cells according to the present invention.

FIG. 3 shows the results of the MTT cytotoxicity assays demonstratingthe efficacy of temozolomide (TMZ) in killing U87, A172 and U251 humanglioma cells.

FIG. 4 shows the results of the MTT cytotoxicity assays demonstratingthe efficacy of the POH-TMZ conjugate in killing U87, A172, and U251human glioma cells according to the present invention.

FIG. 5 shows the results of the MTT cytotoxicity assays demonstratingthe efficacy of the POH-Rolipram conjugate and Rolipram in killing A172human glioma cells.

FIG. 6 shows the results of the MTT cytotoxicity assays demonstratingthe efficacy of the POH-Rolipram conjugate and Rolipram in killing U87human glioma cells.

FIG. 7 shows the results of the MTT cytotoxicity assays demonstratingthe efficacy of the POH-Rolipram conjugate and Rolipram in killing U251human glioma cells.

FIG. 8 shows the results of the MTT cytotoxicity assays demonstratingthe efficacy of the POH-Rolipram conjugate and Rolipram in killing L229human glioma cells.

FIG. 9 shows the inhibition of tumor growth by butyryl-POH in mousemodels. FIG. 9A shows the images of subcutaneous U-87 gliomas in nudemice treated with butyryl-POH, purified (S)-perillyl alcohol having apurity greater than 98.5% (“Purified POH”), POH purchased from Sigmachemicals (“Sigma”), or phosphate buffered saline (“PBS”; negativecontrol). FIG. 9B shows average tumor growth over time (total timeperiod of 60 days).

FIG. 10 shows the results of a Colony forming Assay (CFA) demonstratingthe cytotoxic effect of TMZ and TMZ-POH on TMZ sensitive (U251) and TMZresistant (U251TR) U251 cells.

FIG. 11 shows the results of a Colony forming Assay (CFA) demonstratingthe cytotoxic effect of POH on TMZ sensitive (U251) and TMZ resistant(U251TR) U251 cells.

FIG. 12 shows the results of the MTT cytotoxicity assays demonstratingthe efficacy of the POH-TMZ conjugate in killing U251 cells, U251TRcells, and normal astrocytes.

FIG. 13 shows the results of the MTT cytotoxicity assays demonstratingthe efficacy of the POH-TMZ conjugate in killing normal astrocytes,brain endothelial cells (BEC; confluent and subconfluent), and tumorbrain endothelial cells (TuBEC).

FIG. 14 shows the results of the MTT cytotoxicity assays demonstratingthe efficacy of TMZ and the POH-TMZ conjugate in killing USC-04 gliomacancer stem cells.

FIG. 15 shows the results of the MTT cytotoxicity assays demonstratingthe efficacy of POH in killing USC-04 glioma cancer stem cells.

FIG. 16 shows the results of the MTT cytotoxicity assays demonstratingthe efficacy of TMZ and the POH-TMZ conjugate in killing USC-02 gliomacancer stem cells.

FIG. 17 shows the results of the MTT cytotoxicity assays demonstratingthe efficacy of POH in killing USC-02 glioma cancer stem cells.

FIG. 18 shows a western blot demonstrating that TMZ-POH induces ERstress (ERS) in TMZ sensitive (“U251-TMZs”) and resistant (“U251-TMZr”)U251 glioma cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for a derivative of monoterpene orsesquiterpene, such as a perillyl alcohol derivative. The presentinvention also provides for a pharmaceutical composition comprising aderivative of monoterpene or sesquiterpene, such as a perillyl alcoholderivative. For example, the perillyl alcohol derivative may be aperillyl alcohol carbamate. The perillyl alcohol derivative may beperillyl alcohol conjugated with a therapeutic agent such as achemotherapeutic agent. The monoterpene (or sesquiterpene) derivativemay be formulated into a pharmaceutical composition, where themonoterpene (or sesquiterpene) derivative is present in amounts rangingfrom about 0.01% (w/w) to about 100% (w/w), from about 0.1% (w/w) toabout 80% (w/w), from about 1% (w/w) to about 70% (w/w), from about 10%(w/w) to about 60% (w/w), or from about 0.1% (w/w) to about 20% (w/w).The present compositions can be administered alone, or may beco-administered together with radiation or another agent (e.g., achemotherapeutic agent), to treat a disease such as cancer. Treatmentsmay be sequential, with the monoterpene (or sesquiterpene) derivativebeing administered before or after the administration of other agents.For example, a perillyl alcohol carbamate may be used to sensitize acancer patient to radiation or chemotherapy. Alternatively, agents maybe administered concurrently. The route of administration may vary, andcan include, inhalation, intranasal, oral, transdermal, intravenous,subcutaneous or intramuscular injection. The present invention alsoprovides for a method of treating a disease such as cancer, comprisingthe step of delivering to a patient a therapeutically effective amountof a derivative of monoterpene (or sesquiterpene).

The compositions of the present invention may contain one or more typesof derivatives of monoterpene (or sesquiterpene). Monoterpenes includeterpenes that consist of two isoprene units. Monoterpenes may be linear(acyclic) or contain rings. Derivatives of monoterpenoids are alsoencompassed by the present invention. Monoterpenoids may be produced bybiochemical modifications such as oxidation or rearrangement ofmonoterpenes. Examples of monoterpenes and monoterpenoids include,perillyl alcohol (S(−)) and (R(+)), ocimene, myrcene, geraniol, citral,citronellol, citronellal, linalool, pinene, terpineol, terpinen,limonene, terpinenes, phellandrenes, terpinolene, terpinen-4-ol (or teatree oil), pinene, terpineol, terpinen; the terpenoids such as p-cymenewhich is derived from monocyclic terpenes such as menthol, thymol andcarvacrol; bicyclic monoterpenoids such as camphor, borneol andeucalyptol.

Monoterpenes may be distinguished by the structure of a carbon skeletonand may be grouped into acyclic monoterpenes (e.g., myrcene, (Z)- and(E)-ocimene, linalool, geraniol, nerol, citronellol, myrcenol, geranial,citral a, neral, citral b, citronellal, etc.), monocyclic monoterpenes(e.g., limonene, terpinene, phellandrene, terpinolene, menthol, carveol,etc.), bicyclic monoterpenes (e.g., pinene, myrcenol, myrtenal,verbanol, verbanon, pinocarveol, carene, sabinene, camphene, thujene,etc.) and tricyclic monoterpenes (e.g. tricyclene). See Encyclopedia ofChemical Technology, Fourth Edition, Volume 23, page 834-835.

Sesquiterpenes of the present invention include terpenes that consist ofthree isoprene units. Sesquiterpenes may be linear (acyclic) or containrings. Derivatives of sesquiterpenoids are also encompassed by thepresent invention. Sesquiterpenoids may be produced by biochemicalmodifications such as oxidation or rearrangement of sesquiterpenes.Examples of sesquiterpenes include farnesol, farnesal, farnesylic acidand nerolidol.

The derivatives of monoterpene (or sesquiterpene) include, but are notlimited to, carbamates, esters, ethers, alcohols and aldehydes of themonoterpene (or sesquiterpene). Monoterpene (or sesquiterpene) alcoholsmay be derivatized to carbamates, esters, ethers, aldehydes or acids.

Carbamate refers to a class of chemical compounds sharing the functionalgroup

based on a carbonyl group flanked by an oxygen and a nitrogen. R¹, R²and R³ can be a group such as alkyl, aryl, etc., which can besubstituted. The R groups on the nitrogen and the oxygen may form aring. R¹—OH may be a monoterpene, e.g., POH. The R²—N—R³ moiety may be atherapeutic agent.

Carbamates may be synthesized by reacting isocyanate and alcohol, or byreacting chloroformate with amine. Carbamates may be synthesized byreactions making use of phosgene or phosgene equivalents. For example,carbamates may be synthesized by reacting phosgene gas, diphosgene or asolid phosgene precursor such as triphosgene with two amines or an amineand an alcohol. Carbamates (also known as urethanes) can also be madefrom reaction of a urea intermediate with an alcohol. Dimethyl carbonateand diphenyl carbonate are also used for making carbamates.Alternatively, carbamates may be synthesized through the reaction ofalcohol and/or amine precursors with an ester-substituted diarylcarbonate, such as bismethylsalicylcarbonate (BMSC). U.S. PatentPublication No. 20100113819.

Carbamates may be synthesized by the following approach:

Suitable reaction solvents include, but are not limited to,tetrahydrofuran, dichloromethane, dichloroethane, acetone, anddiisopropyl ether. The reaction may be performed at a temperatureranging from about −70° C. to about 80° C., or from about −65° C. toabout 50° C. The molar ratio of perillyl chloroformate to the substrateR—NH₂ may range from about 1:1 to about 2:1, from about 1:1 to about1.5:1, from about 2:1 to about 1:1, or from about 1.05:1 to about 1.1:1.Suitable bases include, but are not limited to, organic bases, such astriethylamine, potassium carbonate, N,N′-diisopropylethylamine, butyllithium, and potassium-t-butoxide.

Alternatively, carbamates may be synthesized by the following approach:

Suitable reaction solvents include, but are not limited to,dichloromethane, dichloroethane, toluene, diisopropyl ether, andtetrahydrofuran. The reaction may be performed at a temperature rangingfrom about 25° C. to about 110° C., or from about 30° C. to about 80°C., or about 50° C. The molar ratio of perillyl alcohol to the substrateR—N═C═O may range from about 1:1 to about 2:1, from about 1:1 to about1.5:1, from about 2:1 to about 1:1, or from about 1.05:1 to about 1.1:1.

Esters of the monoterpene (or sesquiterpene) alcohols of the presentinvention can be derived from an inorganic acid or an organic acid.Inorganic acids include, but are not limited to, phosphoric acid,sulfuric acid, and nitric acid. Organic acids include, but are notlimited to, carboxylic acid such as benzoic acid, fatty acid, aceticacid and propionic acid, and any therapeutic agent bearing at least onecarboxylic acid functional group Examples of esters of monoterpene (orsesquiterpene) alcohols include, but are not limited to, carboxylic acidesters (such as benzoate esters, fatty acid esters (e.g., palmitateester, linoleate ester, stearate ester, butyryl ester and oleate ester),acetates, propionates (or propanoates), and formates), phosphates,sulfates, and carbamates (e.g., N,N-dimethylaminocarbonyl).Wikipedia—Ester. Retrieved from URL: http://en.wikipedia.org/wiki/Ester.

A specific example of a monoterpene that may be used in the presentinvention is perillyl alcohol (commonly abbreviated as POH). Thederivatives of perillyl alcohol include, perillyl alcohol carbamates,perillyl alcohol esters, perillic aldehydes, dihydroperillic acid,perillic acid, perillic aldehyde derivatives, dihydroperillic acidesters and perillic acid esters. The derivatives of perillyl alcohol mayalso include its oxidative and nucleophilic/electrophilic additionderivatives. U.S. Patent Publication No. 20090031455. U.S. Pat. Nos.6,133,324 and 3,957,856. Many examples of derivatives of perillylalcohol are reported in the chemistry literature (see Appendix A: CASScifinder search output file, retrieved Jan. 25, 2010).

In certain embodiments, a POH carbamate is synthesized by a processcomprising the step of reacting a first reactant of perillylchloroformate with a second reactant such as dimethyl celocoxib (DMC),temozolomide (TMZ) and rolipram. The reaction may be carried out in thepresence of tetrahydrofuran and a base such as n-butyl lithium. Perillylchloroformate may be made by reacting POH with phosgene. For example,POH conjugated with temozolomide through a carbamate bond may besynthesized by reacting temozolomide with oxalyl chloride followed byreaction with perillyl alcohol. The reaction may be carried out in thepresence of 1,2-dichloroethane.

POH carbamates encompassed by the present invention include, but notlimited to, 4-(bis-N,N′-4-isopropenyl cyclohex-1-enylmethyloxycarbonyl[5-(2,5-dimethylphenyl)-3-trifluoromethylpyrazol-1-yl]benzenesulfonamide,4-(3-cyclopentyloxy-4-methoxy phenyl)-2-oxo-pyrrolidine-1-carboxylicacid 4-isopropenyl cyclohex-1-enylmethyl ester, and (3-methyl4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carbonyl)carbamicacid-4-isopropenyl cyclohex-1-enylmethyl ester. The details of thechemical reactions generating these compounds are described in theExamples below.

In certain embodiments, perillyl alcohol derivatives may be perillylalcohol fatty acid esters, such as palmitoyl ester of POH and linoleoylester of POH, the chemical structures of which are shown below.

Hexadecanoic Acid 4-isopropenyl-cyclohex-1-enylmethyl Ester (PalmitoylEster of POH)

Octadeca-9,12-dienoic acid 4-isopropenyl-cyclohex-1-enylmethyl ester(Linoleoyl ester of POH)

The monoterpene (or sesquiterpene) derivative may be a monoterpene (orsesquiterpene) conjugated with a therapeutic agent. A monoterpene (orsesquiterpene) conjugate encompassed by the present invention is amolecule having a monoterpene (or sesquiterpene) covalently bound via achemical linking group to a therapeutic agent. The molar ratio of themonoterpene (or sesquiterpene) to the therapeutic agent in themonoterpene (or sesquiterpene) conjugate may be 1:1, 1:2, 1:3, 1:4, 2:1,3:1, 4:1, or any other suitable molar ratios. The monoterpene (orsesquiterpene) and the therapeutic agent may be covalently linkedthrough carbamate, ester, ether bonds, or any other suitable chemicalfunctional groups. When the monoterpene (or sesquiterpene) and thetherapeutic agent are conjugated through a carbamate bond, thetherapeutic agent may be any agent bearing at least one carboxylic acidfunctional group, or any agent bearing at least one amine functionalgroup. In a specific example, a perillyl alcohol conjugate is perillylalcohol covalently bound via a chemical linking group to achemotherapeutic agent.

According to the present invention, the therapeutic agents that may beconjugated with monoterpene (or sesquiterpene) include, but are notlimited to, chemotherapeutic agents, therapeutic agents for treatment ofCNS disorders (including, without limitation, primary degenerativeneurological disorders such as Alzheimer's, Parkinson's, multiplesclerosis, Attention-Deficit Hyperactivity Disorder or ADHD,psychological disorders, psychosis and depression), immunotherapeuticagents, angiogenesis inhibitors, and anti-hypertensive agents.Anti-cancer agents that may be conjugated with monoterpene orsesquiterpene can have one or more of the following effects on cancercells or the subject: cell death; decreased cell proliferation;decreased numbers of cells; inhibition of cell growth; apoptosis;necrosis; mitotic catastrophe; cell cycle arrest; decreased cell size;decreased cell division; decreased cell survival; decreased cellmetabolism; markers of cell damage or cytotoxicity; indirect indicatorsof cell damage or cytotoxicity such as tumor shrinkage; improvedsurvival of a subject; or disappearance of markers associated withundesirable, unwanted, or aberrant cell proliferation. U.S. PatentPublication No. 20080275057.

Also encompassed by the present invention is admixtures and/orcoformulations of a monoterpene (or sesquiterpene) and at least onetherapeutic agent.

Chemotherapeutic agents include, but are not limited to, DNA alkylatingagents, topoisomerase inhibitors, endoplasmic reticulum stress inducingagents, a platinum compound, an antimetabolite, vincalkaloids, taxanes,epothilones, enzyme inhibitors, receptor antagonists, tyrosine kinaseinhibitors, boron radiosensitizers (i.e. velcade), and chemotherapeuticcombination therapies.

Non-limiting examples of DNA alkylating agents are nitrogen mustards,such as Cyclophosphamide (Ifosfamide, Trofosfamide), Chlorambucil(Melphalan, Prednimustine), Bendamustine, Uramustine and Estramustine;nitrosoureas, such as Carmustine (BCNU), Lomustine (Semustine),Fotemustine, Nimustine, Ranimustine and Streptozocin; alkyl sulfonates,such as Busulfan (Mannosulfan, Treosulfan); Aziridines, such asCarboquone, Triaziquone, Triethylenemelamine; Hydrazines (Procarbazine);Triazenes such as Dacarbazine and Temozolomide (TMZ); Altretamine andMitobronitol.

Non-limiting examples of Topoisomerase I inhibitors include Campothecinderivatives including SN-38, APC, NPC, campothecin, topotecan, exatecanmesylate, 9-nitrocamptothecin, 9-aminocamptothecin, lurtotecan,rubitecan, silatecan, gimatecan, diflomotecan, extatecan, BN-80927,DX-8951f, and MAG-CPT as described in Pommier Y. (2006) Nat. Rev. Cancer6(10):789-802 and U.S. Patent Publication No. 200510250854;Protoberberine alkaloids and derivatives thereof including berberrubineand coralyne as described in Li et al. (2000) Biochemistry39(24):7107-7116 and Gatto et al. (1996) Cancer Res. 15(12):2795-2800;Phenanthroline derivatives including Benzo[i]phenanthridine, Nitidine,and fagaronine as described in Makhey et al. (2003) Bioorg. Med. Chem.11 (8): 1809-1820; Terbenzimidazole and derivatives thereof as describedin Xu (1998) Biochemistry 37(10):3558-3566; and Anthracyclinederivatives including Doxorubicin, Daunorubicin, and Mitoxantrone asdescribed in Foglesong et al. (1992) Cancer Chemother. Pharmacol.30(2):123-]25, Crow et al. (1994) J. Med. Chem. 37(19):31913194, andCrespi et al. (1986) Biochem. Biophys. Res. Commun. 136(2):521-8.Topoisomerase II inhibitors include, but are not limited to Etoposideand Teniposide. Dual topoisomerase I and II inhibitors include, but arenot limited to, Saintopin and other Naphthecenediones, DACA and otherAcridine-4-Carboxamindes, Intoplicine and other Benzopyridoindoles,TAS-103 and other 7H-indeno[2,1-c]Quinoline-7-ones, Pyrazoloacridine, XR11576 and other Benzophenazines, XR 5944 and other Dimeric compounds,7-oxo-7H-dibenz[f,ij]Isoquinolines and 7-oxo-7H-benzo[e]pyrimidines, andAnthracenyl-amino Acid Conjugates as described in Denny and Baguley(2003) Curr. Top. Med. Chem. 3(3):339-353. Some agents inhibitTopoisomerase II and have DNA intercalation activity such as, but notlimited to, Anthracyclines (Aclarubicin, Daunorubicin, Doxorubicin,Epirubicin, Idarubicin, Amrubicin, Pirarubicin, Valrubicin, Zorubicin)and Antracenediones (Mitoxantrone and Pixantrone).

Examples of endoplasmic reticulum stress inducing agents include, butare not limited to, dimethyl-celecoxib (DMC), nelfinavir, celecoxib, andboron radiosensitizers (i.e. velcade (Bortezomib)).

Platinum based compounds are a subclass of DNA alkylating agents.Non-limiting examples of such agents include Cisplatin, Nedaplatin,Oxaliplatin, Triplatin tetranitrate, Satraplatin, Aroplatin, Lobaplatin,and JM-216. (see McKeage et al. (1997) J. Clin. Oncol. 201: 1232-1237and in general, CHEMOTHERAPY FOR GYNECOLOGICAL NEOPLASM, CURRENT THERAPYAND NOVEL APPROACHES, in the Series Basic and Clinical Oncology, Angioliet al. Eds., 2004).

“FOLFOX” is an abbreviation for a type of combination therapy that isused to treat colorectal cancer. It includes 5-FU, oxaliplatin andleucovorin. Information regarding this treatment is available on theNational Cancer Institute's web site, cancer.gov, last accessed on Jan.16, 2008.

“FOLFOX/BV” is an abbreviation for a type of combination therapy that isused to treat colorectal cancer. This therapy includes 5-FU,oxaliplatin, leucovorin and Bevacizumab. Furthermore, “XELOX/BV” isanother combination therapy used to treat colorectal cancer, whichincludes the prodrug to 5-FU, known as Capecitabine (Xeloda) incombination with oxaliplatin and bevacizumab. Information regardingthese treatments are available on the National Cancer Institute's website, cancer.gov or from 23 the National Comprehensive Cancer Network'sweb site, nccn.org, last accessed on May 27, 2008.

Non-limiting examples of antimetabolite agents include Folic acid based,i.e. dihydrofolate reductase inhibitors, such as Aminopterin,Methotrexate and Pemetrexed; thymidylate synthase inhibitors, such asRaltitrexed, Pemetrexed; Purine based, i.e. an adenosine deaminaseinhibitor, such as Pentostatin, a thiopurine, such as Thioguanine andMercaptopurine, a halogenated/ribonucleotide reductase inhibitor, suchas Cladribine, Clofarabine, Fludarabine, or a guanine/guanosine:thiopurine, such as Thioguanine; or Pyrimidine based, i.e.cytosine/cytidine: hypomethylating agent, such as Azacitidine andDecitabine, a DNA polymerase inhibitor, such as Cytarabine, aribonucleotide reductase inhibitor, such as Gemcitabine, or athymine/thymidine: thymidylate synthase inhibitor, such as aFluorouracil (5-FU). Equivalents to 5-FU include prodrugs, analogs andderivative thereof such as 5′-deoxy-5-fluorouridine (doxifluoroidine),1-tetrahydrofuranyl-5-fluorouracil (ftorafur), Capecitabine (Xeloda),S-I (MBMS-247616, consisting of tegafur and two modulators, a5-chloro-2,4-dihydroxypyridine and potassium oxonate), ralititrexed(tomudex), nolatrexed (Thymitaq, AG337), LY231514 and ZD9331, asdescribed for example in Papamicheal (1999) The Oncologist 4:478-487.

Examples of vincalkaloids, include, but are not limited to Vinblastine,Vincristine, Vinflunine, Vindesine and Vinorelbine.

Examples of taxanes include, but are not limited to docetaxel,Larotaxel, Ortataxel, Paclitaxel and Tesetaxel. An example of anepothilone is iabepilone.

Examples of enzyme inhibitors include, but are not limited tofarnesyltransferase inhibitors (Tipifarnib); CDK inhibitor (Alvocidib,Seliciclib); proteasome inhibitor (Bortezomib); phosphodiesteraseinhibitor (Anagrelide; rolipram); IMP dehydrogenase inhibitor(Tiazofurine); and lipoxygenase inhibitor (Masoprocol). Examples ofreceptor antagonists include, but are not limited to ERA (Atrasentan);retinoid X receptor (Bexarotene); and a sex steroid (Testolactone).

Examples of tyrosine kinase inhibitors include, but are not limited toinhibitors to ErbB: HER1/EGFR (Erlotinib, Gefitinib, Lapatinib,Vandetanib, Sunitinib, Neratinib); HER2/neu (Lapatinib, Neratinib); RTKclass III: C-kit (Axitinib, Sunitinib, Sorafenib), FLT3 (Lestaurtinib),PDGFR (Axitinib, Sunitinib, Sorafenib); and VEGFR (Vandetanib,Semaxanib, Cediranib, Axitinib, Sorafenib); bcr-abl (Imatinib,Nilotinib, Dasatinib); Src (Bosutinib) and Janus kinase 2(Lestaurtinib).

“Lapatinib” (Tykerb®) is an dual EGFR and erbB-2 inhibitor. Lapatinibhas been investigated as an anticancer monotherapy, as well as incombination with trastuzumab, capecitabine, letrozole, paclitaxel andFOLFIRI (irinotecan, 5-fluorouracil and leucovorin), in a number ofclinical trials. It is currently in phase III testing for the oraltreatment of metastatic breast, head and neck, lung, gastric, renal andbladder cancer.

A chemical equivalent of lapatinib is a small molecule or compound thatis a tyrosine kinase inhibitor (TKI) or alternatively a HER-1 inhibitoror a HER-2 inhibitor. Several TKIs have been found to have effectiveantitumor activity and have been approved or are in clinical trials.Examples of such include, but are not limited to, Zactima (ZD6474),Iressa (gefitinib), imatinib mesylate (STI571; Gleevec), erlotinib(OSI-1774; Tarceva), canertinib (CI-1033), semaxinib (SU5416), vatalanib(PTK787/ZK222584), sorafenib (BAY 43-9006), sutent (SUI 1248) andlefltmomide (SU101).

PTK/ZK is a tyrosine kinase inhibitor with broad specificity thattargets all VEGF receptors (VEGFR), the platelet-derived growth factor(PDGF) receptor, c-KIT and c-Fms. Drevs (2003) Idrugs 6(8):787-794.PTK/ZK is a targeted drug that blocks angiogenesis and lymphangiogenesisby inhibiting the activity of all known receptors that bind VEGFincluding VEGFR-I (Flt-1), VEGFR-2 (KDR/Flk-1) and VEGFR-3 (Flt-4). Thechemical names of PTK/ZK are1-[4-Chloroanilino]-4-[4-pyridylmethyl]phthalazine Succinate or1-Phthalazinamine, N-(4-chlorophenyl)-4-(4-pyridinylmethyl)-butanedioate(1:1). Synonyms and analogs of PTK/TK are known as Vatalanib, CGP79787D,PTK787/ZK 222584, CGP-79787, DE-00268, PTK-787, PTK787A, VEGFR-TKinhibitor, ZK 222584 and ZK.

Chemotherapeutic agents that can be conjugated with monoterpene orsesquiterpene may also include amsacrine, Trabectedin, retinoids(Alitretinoin, Tretinoin), Arsenic trioxide, asparagine depleterAsparaginase/Pegaspargase), Celecoxib, Demecolcine, Elesclomol,Elsamitrucin, Etoglucid, Lonidamine, Lucanthone, Mitoguazone, Mitotane,Oblimersen, Temsirolimus, and Vorinostat.

The monoterpene or sesquiterpene derivative may be conjugated withangiogenesis inhibitors. Examples of angiogenesis inhibitors include,but are not limited to, angiostatin, angiozyme, antithrombin III,AG3340, VEGF inhibitors, batimastat, bevacizumab (avastin), BMS-275291,CAI, 2C3, HuMV833 Canstatin, Captopril, carboxyamidotriazole, cartilagederived inhibitor (CDI), CC-5013,6-O-(chloroacetyl-carbonyl)-fumagillol, COL-3, combretastatin,combretastatin A4 Phosphate, Dalteparin, EMD 121974 (Cilengitide),endostatin, erlotinib, gefitinib (Iressa), genistein, halofuginonehydrobromide, Id1, Id3, IM862, imatinib mesylate, IMC-IC11 Inducibleprotein 10, interferon-alpha, interleukin 12, lavendustin A, LY317615 orAE-941, marimastat, mspin, medroxpregesterone acetate, Meth-1,Meth-2,2-methoxyestradiol (2-ME), neovastat, oteopontin cleaved product,PEX, pigment epithelium growth factor (PEGF), platelet factor 4,prolactin fragment, proliferin-related protein (PRP), PTK787/ZK 222584,ZD6474, recombinant human platelet factor 4 (rPF4), restin, squalamine,SU5416, SU6668, SU11248 suramin, Taxol, Tecogalan, thalidomide,thrombospondin, TNP-470, troponin-1, vasostatin, VEG1, VEGF-Trap, andZD6474.

Non-limiting examples of angiogenesis inhibitors also include, tyrosinekinase inhibitors, such as inhibitors of the tyrosine kinase receptorsFlt-1 (VEGFR1) and Flk-1/KDR (VEGFR2), inhibitors of epidermal-derived,fibroblast-derived, or platelet derived growth factors, MMP (matrixmetalloprotease) inhibitors, integrin blockers, pentosan polysulfate,angiotensin II antagonists, cyclooxygenase inhibitors (includingnon-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin andibuprofen, as well as selective cyclooxygenase-2 inhibitors such ascelecoxib and rofecoxib), and steroidal anti-inflammatories (such ascorticosteroids, mineralocorticoids, dexamethasone, prednisone,prednisolone, methylpred, betamethasone).

Other therapeutic agents that modulate or inhibit angiogenesis and mayalso be conjugated with monoterpene or sesquiterpene include agents thatmodulate or inhibit the coagulation and fibrinolysis systems, including,but not limited to, heparin, low molecular weight heparins andcarboxypeptidase U inhibitors (also known as inhibitors of activethrombin activatable fibrinolysis inhibitor [TAFIa]). U.S. PatentPublication No. 20090328239. U.S. Pat. No. 7,638,549.

Non-limiting examples of the anti-hypertensive agents includeangiotensin converting enzyme inhibitors (e.g., captopril, enalapril,delapril etc.), angiotensin II antagonists (e.g., candesartan cilexetil,candesartan, losartan (or Cozaar), losartan potassium, eprosartan,valsartan (or Diovan), termisartan, irbesartan, tasosartan, olmesartan,olmesartan medoxomil etc.), calcium antagonists (e.g., manidipine,nifedipine, amlodipine (or Amlodin), efonidipine, nicardipine etc.),diuretics, renin inhibitor (e.g., aliskiren etc.), aldosteroneantagonists (e.g., spironolactone, eplerenone etc.), beta-blockers(e.g., metoprolol (or Toporol), atenolol, propranolol, carvedilol,pindolol etc.), vasodilators (e.g., nitrate, soluble guanylate cyclasestimulator or activator, prostacycline etc.), angiotensin vaccine,clonidine and the like. U.S. Patent Publication No. 20100113780.

Other therapeutic agents that may be conjugated with monoterpene (orsesquiterpene) include, but are not limited to, Sertraline (Zoloft),Topiramate (Topamax), Duloxetine (Cymbalta), Sumatriptan (Imitrex),Pregabalin (Lyrica), Lamotrigine (Lamictal), Valaciclovir (Valtrex),Tamsulosin (Flomax), Zidovudine (Combivir), Lamivudine (Combivir),Efavirenz (Sustiva), Abacavir (Epzicom), Lopinavir (Kaletra),Pioglitazone (Actos), Desloratidine (Clarinex), Cetirizine (Zyrtec),Pentoprazole (Protonix), Lansoprazole (Prevacid), Rebeprazole (Aciphex),Moxifloxacin (Avelox), Meloxicam (Mobic), Dorzolamide (Truspot),Diclofenac (Voltaren), Enlapril (Vasotec), Montelukast (Singulair),Sildenafil (Viagra), Carvedilol (Coreg), Ramipril (Delix).

Table 1 lists pharmaceutical agents that can be conjugated withmonoterpene (or sesquiterpene), including structure of thepharmaceutical agent and the preferred derivative for conjugation.

TABLE 1 Brand Generic Preferred Name Name Activity Structure DerivativeZoloft Sertraline Depression

Carbamate Topamax Topiramate Seizures

Carbamate Cymbalta Duloxetine Depression

Carbamate Imitrex Sumatriptan Migraine

Carbamate Lyrica Pregabalin Neuropathic pain

Carbamate or Ester Lamictal Lamotrigine Seizures

Carbamate Valtrex Valaciclovir Herpes

Carbamate Tarceva Erlotinib Non-small cell lung cancer

Carbamate Flomax Tamsulosin Benign prostatic Cancer

Carbamate Gleevec Imatinib Leukemia

Carbamate Combivir Zidovudine HIV infection

Carbamate Combivir Lamivudine HIV infection

Carbamate Sustiva Efavirenz HIV infection

Carbamate Epzicom Abacavir HIV infection

Carbamate Kaletra Lopinavir HIV infection

Carbamate Actos Pioglitazone Type-2 diabetes

Carbamate Clarinex Desloratidine Allergic rhinitis

Carbamate Zyrtec Cetirizine Allergic

Ester Protonix Pentoprazole Gastro- intestinal

Carbamate Prevacid Lansoprazole Gastro- intestinal

Carbamate Aciphex Rebeprazole Gastro- intestinal

Carbamate Diovan Valsartan Hypertension

Carbamate Cozaar Losartan Hypertension

Carbamate Avelox Moxifloxacin Bacterial infection

Carbamate or Ester Mobic Meloxicam Osteoarthritis

Carbamate Truspot Dorzolamide Intraocular pressure

Carbamate Voltaren Diclofenac Osteoarthritis & rheumatoid arthritis

Carbamate or Ester Vasotec Enlapril Hypertension

Carbamate or Ester Singulair Montelukast Asthma

Ester Amlodin Amlodipine Hypertension

Carbamate Toporol Metoprolol Hypertension

Carbamate Viagra Sildenafil Erectile dysfunction

Carbamate Coreg Carvedilol Hypertension

Carbamate Delix Ramipril Hypertension

Carbamate or Ester Sinemet (Parcopa, Atamet) L-DOPA Neurologicaldisorders

The purity of the monoterpene (or sesquiterpene) derivatives may beassayed by gas chromatography (GC) or high pressure liquidchromatography (HPLC). Other techniques for assaying the purity ofmonoterpene (or sesquiterpene) derivatives and for determining thepresence of impurities include, but are not limited to, nuclear magneticresonance (NMR) spectroscopy, mass spectrometry (MS), GC-MS, infraredspectroscopy (IR), and thin layer chromatography (TLC). Chiral puritycan be assessed by chiral GC or measurement of optical rotation.

The monoterpene (or sesquiterpene) derivatives may be purified bymethods such as crystallization, or by separating the monoterpene (orsesquiterpene) derivative from impurities according to the uniquephysicochemical properties (e.g., solubility or polarity) of thederivative. Accordingly, the monoterpene (or sesquiterpene) derivativecan be separated from the monoterpene (or sesquiterpene) by suitableseparation techniques known in the art, such as preparativechromatography, (fractional) distillation, or (fractional)crystallization.

The invention also provides for methods of using monoterpenes (orsesquiterpenes) derivatives to treat a disease, such as cancer or othernervous system disorders. A monoterpenes (or sesquiterpenes) derivativemay be administered alone, or in combination with radiation, surgery orchemotherapeutic agents. A monoterpene or sesquiterpene derivative mayalso be co-administered with antiviral agents, anti-inflammatory agentsor antibiotics. The agents may be administered concurrently orsequentially. A monoterpenes (or sesquiterpenes) derivative can beadministered before, during or after the administration of the otheractive agent(s).

The monoterpene or sesquiterpene derivative may be used in combinationwith radiation therapy. In one embodiment, the present inventionprovides for a method of treating tumor cells, such as malignant gliomacells, with radiation, where the cells are treated with an effectiveamount of a monoterpene derivative, such as a perillyl alcoholcarbamate, and then exposed to radiation. Monoterpene derivativetreatment may be before, during and/or after radiation. For example, themonoterpene or sesquiterpene derivative may be administered continuouslybeginning one week prior to the initiation of radiotherapy and continuedfor two weeks after the completion of radiotherapy. U.S. Pat. Nos.5,587,402 and 5,602,184.

In one embodiment, the present invention provides for a method oftreating tumor cells, such as malignant glioma cells, with chemotherapy,where the cells are treated with an effective amount of a monoterpenederivative, such as a perillyl alcohol carbamate, and then exposed tochemotherapy. Monoterpene derivative treatment may be before, duringand/or after chemotherapy.

Monoterpene (or sesquiterpene) derivatives may be used for the treatmentof nervous system cancers, such as a malignant glioma (e.g.,astrocytoma, anaplastic astrocytoma, glioblastoma multiforme),retinoblastoma, pilocytic astrocytomas (grade I), meningiomas,metastatic brain tumors, neuroblastoma, pituitary adenomas, skull basemeningiomas, and skull base cancer. As used herein, the term “nervoussystem tumors” refers to a condition in which a subject has a malignantproliferation of nervous system cells.

Cancers that can be treated by the present monoterpene (orsesquiterpene) derivatives include, but are not limited to, lung cancer,ear, nose and throat cancer, leukemia, colon cancer, melanoma,pancreatic cancer, mammary cancer, prostate cancer, breast cancer,hematopoietic cancer, ovarian cancer, basal cell carcinoma, biliarytract cancer; bladder cancer; bone cancer; breast cancer; cervicalcancer; choriocarcinoma; colon and rectum cancer; connective tissuecancer; cancer of the digestive system; endometrial cancer; esophagealcancer; eye cancer; cancer of the head and neck; gastric cancer;intra-epithelial neoplasm; kidney cancer; larynx cancer; leukemiaincluding acute myeloid leukemia, acute lymphoid leukemia, chronicmyeloid leukemia, chronic lymphoid leukemia; liver cancer; lymphomaincluding Hodgkin's and Non-Hodgkin's lymphoma; myeloma; fibroma,neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, andpharynx); ovarian cancer; pancreatic cancer; prostate cancer;retinoblastoma; rhabdomyosarcoma; rectal cancer; renal cancer; cancer ofthe respiratory system; sarcoma; skin cancer; stomach cancer; testicularcancer; thyroid cancer; uterine cancer; cancer of the urinary system, aswell as other carcinomas and sarcomas. U.S. Pat. No. 7,601,355.

The present invention also provides methods of treating CNS disorders,including, without limitation, primary degenerative neurologicaldisorders such as Alzheimer's, Parkinson's, psychological disorders,psychosis and depression. Treatment may consist of the use of amonoterpene or sesquiterpene derivative alone or in combination withcurrent medications used in the treatment of Parkinson's, Alzheimer's,or psychological disorders.

The present invention also provides a method of improvingimmunomodulatory therapy responses comprising the steps of exposingcells to an effective amount of a monoterpene or sisquiterpenederivative, such as a perillyl alcohol carbamate, before or duringimmunomodulatory treatment. Preferred immunomodulatory agents arecytokines, such interleukins, lymphokines, monokines, interfereons andchemokines.

The present composition may be administered by any method known in theart, including, without limitation, intranasal, oral, transdermal,ocular, intraperitoneal, inhalation, intravenous, ICV, intracisternalinjection or infusion, subcutaneous, implant, vaginal, sublingual,urethral (e.g., urethral suppository), subcutaneous, intramuscular,intravenous, rectal, sub-lingual, mucosal, ophthalmic, spinal,intrathecal, intra-articular, intra-arterial, sub-arachinoid, bronchialand lymphatic administration. Topical formulation may be in the form ofgel, ointment, cream, aerosol, etc; intranasal formulation can bedelivered as a spray or in a drop; transdermal formulation may beadministered via a transdermal patch or iontorphoresis; inhalationformulation can be delivered using a nebulizer or similar device.Compositions can also take the form of tablets, pills, capsules,semisolids, powders, sustained release formulations, solutions,suspensions, elixirs, aerosols, or any other appropriate compositions.

To prepare such pharmaceutical compositions, one or more of monoterpene(or sesquiterpene) derivatives may be mixed with a pharmaceuticalacceptable carrier, adjuvant and/or excipient, according to conventionalpharmaceutical compounding techniques. Pharmaceutically acceptablecarriers that can be used in the present compositions encompass any ofthe standard pharmaceutical carriers, such as a phosphate bufferedsaline solution, water, and emulsions, such as an oil/water or water/oilemulsion, and various types of wetting agents. The compositions canadditionally contain solid pharmaceutical excipients such as starch,cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, magnesium stearate, sodium stearate, glycerolmonostearate, sodium chloride, dried skim milk and the like. Liquid andsemisolid excipients may be selected from glycerol, propylene glycol,water, ethanol and various oils, including those of petroleum, animal,vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineraloil, sesame oil, etc. Liquid carriers, particularly for injectablesolutions, include water, saline, aqueous dextrose, and glycols. Forexamples of carriers, stabilizers and adjuvants, see Remington'sPharmaceutical Sciences, edited by E. W. Martin (Mack PublishingCompany, 18th ed., 1990). The compositions also can include stabilizersand preservatives.

As used herein, the term “therapeutically effective amount” is an amountsufficient to treat a specified disorder or disease or alternatively toobtain a pharmacological response treating a disorder or disease.Methods of determining the most effective means and dosage ofadministration can vary with the composition used for therapy, thepurpose of the therapy, the target cell being treated, and the subjectbeing treated. Treatment dosages generally may be titrated to optimizesafety and efficacy. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician. Suitable dosage formulations and methods of administering theagents can be readily determined by those of skill in the art. Forexample, the composition are administered at about 0.01 mg/kg to about200 mg/kg, about 0.1 mg/kg to about 100 mg/kg, or about 0.5 mg/kg toabout 50 mg/kg. When the compounds described herein are co-administeredwith another agent or therapy, the effective amount may be less thanwhen the agent is used alone.

Transdermal formulations may be prepared by incorporating the activeagent in a thixotropic or gelatinous carrier such as a cellulosicmedium, e.g., methyl cellulose or hydroxyethyl cellulose, with theresulting formulation then being packed in a transdermal device adaptedto be secured in dermal contact with the skin of a wearer. If thecomposition is in the form of a gel, the composition may be rubbed ontoa membrane of the patient, for example, the skin, preferably intact,clean, and dry skin, of the shoulder or upper arm and or the uppertorso, and maintained thereon for a period of time sufficient fordelivery of the monoterpene (or sesquiterpene) derivative to the bloodserum of the patient. The composition of the present invention in gelform may be contained in a tube, a sachet, or a metered pump. Such atube or sachet may contain one unit dose, or more than one unit dose, ofthe composition. A metered pump may be capable of dispensing one metereddose of the composition.

This invention also provides the compositions as described above forintranasal administration. As such, the compositions can furthercomprise a permeation enhancer. Southall et al. Developments in NasalDrug Delivery, 2000. The monoterpene (or sesquiterpene) derivative maybe administered intranasally in a liquid form such as a solution, anemulsion, a suspension, drops, or in a solid form such as a powder, gel,or ointment. Devices to deliver intranasal medications are well known inthe art. Nasal drug delivery can be carried out using devices including,but not limited to, intranasal inhalers, intranasal spray devices,atomizers, nasal spray bottles, unit dose containers, pumps, droppers,squeeze bottles, nebulizers, metered dose inhalers (MDI), pressurizeddose inhalers, insufflators, and bi-directional devices. The nasaldelivery device can be metered to administer an accurate effectivedosage amount to the nasal cavity. The nasal delivery device can be forsingle unit delivery or multiple unit delivery. In a specific example,the ViaNase Electronic Atomizer from Kurve Technology (Bethell, Wash.)can be used in this invention (http://www.kurvetech.com). The compoundsof the present invention may also be delivered through a tube, acatheter, a syringe, a packtail, a pledget, a nasal tampon or bysubmucosal infusion. U.S. Patent Publication Nos. 20090326275,20090291894, 20090281522 and 20090317377.

The monoterpene (or sesquiterpene) derivative can be formulated asaerosols using standard procedures. The monoterpene (or sesquiterpene)derivative may be formulated with or without solvents, and formulatedwith or without carriers. The formulation may be a solution, or may bean aqueous emulsion with one or more surfactants. For example, anaerosol spray may be generated from pressurized container with asuitable propellant such as, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, hydrocarbons,compressed air, nitrogen, carbon dioxide, or other suitable gas. Thedosage unit can be determined by providing a valve to deliver a meteredamount. Pump spray dispensers can dispense a metered dose or a dosehaving a specific particle or droplet size. As used herein, the term“aerosol” refers to a suspension of fine solid particles or liquidsolution droplets in a gas. Specifically, aerosol includes a gas-bornesuspension of droplets of a monoterpene (or sesquiterpene), as may beproduced in any suitable device, such as an MDI, a nebulizer, or a mistsprayer. Aerosol also includes a dry powder composition of thecomposition of the instant invention suspended in air or other carriergas. Gonda (1990) Critical Reviews in Therapeutic Drug Carrier Systems6:273-313. Raeburn et al., (1992) Pharmacol. Toxicol. Methods27:143-159.

The monoterpene (or sesquiterpene) derivative may be delivered to thenasal cavity as a powder in a form such as microspheres delivered by anasal insufflator. The monoterpene (or sesquiterpene) derivative may beabsorbed to a solid surface, for example, a carrier. The powder ormicrospheres may be administered in a dry, air-dispensable form. Thepowder or microspheres may be stored in a container of the insufflator.Alternatively the powder or microspheres may be filled into a capsule,such as a gelatin capsule, or other single dose unit adapted for nasaladministration.

The pharmaceutical composition can be delivered to the nasal cavity bydirect placement of the composition in the nasal cavity, for example, inthe form of a gel, an ointment, a nasal emulsion, a lotion, a cream, anasal tampon, a dropper, or a bioadhesive strip. In certain embodiments,it can be desirable to prolong the residence time of the pharmaceuticalcomposition in the nasal cavity, for example, to enhance absorption.Thus, the pharmaceutical composition can optionally be formulated with abioadhesive polymer, a gum (e.g., xanthan gum), chitosan (e.g., highlypurified cationic polysaccharide), pectin (or any carbohydrate thatthickens like a gel or emulsifies when applied to nasal mucosa), amicrosphere (e.g., starch, albumin, dextran, cyclodextrin), gelatin, aliposome, carbamer, polyvinyl alcohol, alginate, acacia, chitosansand/or cellulose (e.g., methyl or propyl; hydroxyl or carboxy;carboxymethyl or hydroxylpropyl).

The composition containing the purified monoterpene (or sesquiterpene)can be administered by oral inhalation into the respiratory tract, i.e.,the lungs.

Typical delivery systems for inhalable agents include nebulizerinhalers, dry powder inhalers (DPI), and metered-dose inhalers (MDI).

Nebulizer devices produce a stream of high velocity air that causes atherapeutic agent in the form of liquid to spray as a mist. Thetherapeutic agent is formulated in a liquid form such as a solution or asuspension of particles of suitable size. In one embodiment, theparticles are micronized. The term “micronized” is defined as havingabout 90% or more of the particles with a diameter of less than about 10μm. Suitable nebulizer devices are provided commercially, for example,by PARI GmbH (Starnberg, Germany). Other nebulizer devices includeRespimat (Boehringer Ingelheim) and those disclosed in, for example,U.S. Pat. Nos. 7,568,480 and 6,123,068, and WO 97/12687. Themonoterpenes (or sesquiterpenes) can be formulated for use in anebulizer device as an aqueous solution or as a liquid suspension.

DPI devices typically administer a therapeutic agent in the form of afree flowing powder that can be dispersed in a patient's air-streamduring inspiration. DPI devices which use an external energy source mayalso be used in the present invention. In order to achieve a freeflowing powder, the therapeutic agent can be formulated with a suitableexcipient (e.g., lactose). A dry powder formulation can be made, forexample, by combining dry lactose having a particle size between about 1μm and 100 μm with micronized particles of the monoterpenes (orsesquiterpenes) and dry blending. Alternatively, the monoterpene can beformulated without excipients. The formulation is loaded into a drypowder dispenser, or into inhalation cartridges or capsules for use witha dry powder delivery device. Examples of DPI devices providedcommercially include Diskhaler (GlaxoSmithKline, Research Triangle Park,N.C.) (see, e.g., U.S. Pat. No. 5,035,237); Diskus (GlaxoSmithKline)(see, e.g., U.S. Pat. No. 6,378,519; Turbuhaler (AstraZeneca,Wilmington, Del.) (see, e.g., U.S. Pat. No. 4,524,769); and Rotahaler(GlaxoSmithKline) (see, e.g., U.S. Pat. No. 4,353,365). Further examplesof suitable DPI devices are described in U.S. Pat. Nos. 5,415,162,5,239,993, and 5,715,810 and references therein.

MDI devices typically discharge a measured amount of therapeutic agentusing compressed propellant gas. Formulations for MDI administrationinclude a solution or suspension of active ingredient in a liquefiedpropellant. Examples of propellants include hydrofluoroalklanes (HFA),such as 1,1,1,2-tetrafluoroethane (HFA 134a) and1,1,1,2,3,3,3-heptafluoro-n-propane, (HFA 227), and chlorofluorocarbons,such as CCl₃F. Additional components of HFA formulations for MDIadministration include co-solvents, such as ethanol, pentane, water; andsurfactants, such as sorbitan trioleate, oleic acid, lecithin, andglycerin. (See, for example, U.S. Pat. No. 5,225,183, EP 0717987, and WO92/22286). The formulation is loaded into an aerosol canister, whichforms a portion of an MDI device. Examples of MDI devices developedspecifically for use with HFA propellants are provided in U.S. Pat. Nos.6,006,745 and 6,143,227. For examples of processes of preparing suitableformulations and devices suitable for inhalation dosing see U.S. Pat.Nos. 6,268,533, 5,983,956, 5,874,063, and 6,221,398, and WO 99/53901, WO00/61108, WO 99/55319 and WO 00/30614.

The monoterpene (or sesquiterpene) derivative may be encapsulated inliposomes or microcapsules for delivery via inhalation. A liposome is avesicle composed of a lipid bilayer membrane and an aqueous interior.The lipid membrane may be made of phospholipids, examples of whichinclude phosphatidylcholine such as lecithin and lysolecithin; acidicphospholipids such as phosphatidylserine and phosphatidylglycerol; andsphingophospholipids such as phosphatidylethanolamine and sphingomyelin.Alternatively, cholesterol may be added. A microcapsule is a particlecoated with a coating material. For example, the coating material mayconsist of a mixture of a film-forming polymer, a hydrophobicplasticizer, a surface activating agent or/and a lubricantnitrogen-containing polymer. U.S. Pat. Nos. 6,313,176 and 7,563,768.

The monoterpene (or sesquiterpene) derivative may also be used alone orin combination with other chemotherapeutic agents via topicalapplication for the treatment of localized cancers such as breast canceror melanomas. The monoterpene (or sesquiterpene) derivative may also beused in combination with narcotics or analgesics for transdermaldelivery of pain medication.

This invention also provides the compositions as described above forocular administration. As such, the compositions can further comprise apermeation enhancer. For ocular administration, the compositionsdescribed herein can be formulated as a solution, emulsion, suspension,etc. A variety of vehicles suitable for administering compounds to theeye are known in the art. Specific non-limiting examples are describedin U.S. Pat. Nos. 6,261,547; 6,197,934; 6,056,950; 5,800,807; 5,776,445;5,698,219; 5,521,222; 5,403,841; 5,077,033; 4,882,150; and 4,738,851.

The monoterpene (or sesquiterpene) derivative can be given alone or incombination with other drugs for the treatment of the above diseases fora short or prolonged period of time. The present compositions can beadministered to a mammal, preferably a human. Mammals include, but arenot limited to, murines, rats, rabbit, simians, bovines, ovine, porcine,canines, feline, farm animals, sport animals, pets, equine, andprimates.

The invention also provides a method for inhibiting the growth of a cellin vitro, ex vivo or in vivo, where a cell, such as a cancer cell, iscontacted with an effective amount of the monoterpene (or sesquiterpene)derivative as described herein.

Pathological cells or tissue such as hyperproliferative cells or tissuemay be treated by contacting the cells or tissue with an effectiveamount of a composition of this invention. The cells, such as cancercells, can be primary cancer cells or can be cultured cells availablefrom tissue banks such as the American Type Culture Collection (ATCC).The pathological cells can be cells of a systemic cancer, gliomas,meningiomas, pituitary adenomas, or a CNS metastasis from a systemiccancer, lung cancer, prostate cancer, breast cancer, hematopoieticcancer or ovarian cancer. The cells can be from a vertebrate, preferablya mammal, more preferably a human. U.S. Patent Publication No.2004/0087651. Balassiano et al. (2002) Intern. J. Mol. Med. 10:785-788.Thorne, et al. (2004) Neuroscience 127:481-496. Fernandes, et al. (2005)Oncology Reports 13:943-947. Da Fonseca, et al. (2008) SurgicalNeurology 70:259267. Da Fonseca, et al. (2008) Arch. Immunol. Ther. Exp.56:267-276. Hashizume, et al. (2008) Neuroncology 10:112-120.

In vitro efficacy of the present composition can be determined usingmethods well known in the art. For example, the cytoxicity of thepresent monoterpene (or sesquiterpene) and/or the therapeutic agents maybe studied by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide]cytotoxicity assay. MTT assay is based on the principle ofuptake of MTT, a tetrazolium salt, by metabolically active cells whereit is metabolized into a blue colored formazon product, which can beread spectrometrically. J. of Immunological Methods 65: 55 63, 1983. Thecytoxicity of the present monoterpene (or sesquiterpene) derivativeand/or the therapeutic agents may be studied by colony formation assay.Functional assays for inhibition of VEGF secretion and IL-8 secretionmay be performed via ELISA. Cell cycle block by the present monoterpene(or sesquiterpene) derivative and/or the therapeutic agents may bestudied by standard propidium iodide (PI) staining and flow cytometry.Invasion inhibition may be studied by Boyden chambers. In this assay alayer of reconstituted basement membrane, Matrigel, is coated ontochemotaxis filters and acts as a barrier to the migration of cells inthe Boyden chambers. Only cells with invasive capacity can cross theMatrigel barrier. Other assays include, but are not limited to cellviability assays, apoptosis assays, and morphological assays.

The following are examples of the present invention and are not to beconstrued as limiting.

EXAMPLES Example 1 Synthesis of Dimethyl Celecoxib bisPOH Carbamate(4-(bis-N,N′-4-isopropenyl cyclohex-1-enylmethyloxycarbonyl[5-(2,5-dimethylphenyl)-3-trifluoromethylpyrazol-1-yl]benzenesulfonamide)

The reaction scheme is the following:

Phosgene (20% in toluene, 13 ml, 26.2 mmol) was added to a mixture ofperillyl alcohol (2.0 grams, 13.1 mmol) and potassium carbonate (5.4grams, 39.1 mmol) in dry toluene (30 mL) over a period of 30 minuteswhile maintaining the temperature between 10° C. to 15° C. The reactionmixture was allowed to warm to room temperature and stirred for 8.0hours under N₂. The reaction mixture was quenched with water (30 mL) andthe organic layer was separated. The aqueous layer was extracted withtoluene (20 mL) and the combined organic layer was washed with water (50mL×2), brine (15%, 30 mL) and dried over sodium sulfate (20 grams). Thefiltered organic layer was concentrated under vacuum to give perillylchloroformate as an oil. Weight: 2.5 grams; Yield: 89%. ¹H-NMR (400 MHz,CDCl₃): δ 1.5 (m, 1H), 1.7 (s, 3H), 1.8 (m, 1H), 2.0 (m, 1H), 2.2 (m,4H), 4.7 (dd, 4H); 5.87 (m, 1H).

Perillyl chloroformate (0.11 grams, 0.55 mmol) was added slowly to amixture of dimethyl celecoxib (0.2 grams, 0.50 mmol) and potassiumcarbonate (0.13 grams, 1.0 mmol) in dry acetone (10 mL) over a period of5 minutes under N₂. The reaction mixture was heated to reflux andmaintained for 3 hours. Since TLC analysis indicated the presence ofdimethyl celecoxib (>60%), another 1.0 equivalent of perillylchloroformate was added and refluxed for an additional 5 hours. Thereaction mixture was cooled and acetone was concentrated under vacuum togive a residue.

The resulting residue was suspended in water (15 mL) and extracted withethyl acetate (3×15 mL). The combined organic layer was washed withwater (20 mL) followed by brine (15%, 20 mL) and dried over sodiumsulfate. The filtered organic layer was concentrated under vacuum togive a residue which was purified by column chromatography [columndimensions: diameter: 1.5 cm, height: 10 cm, silica: 230-400 mesh] andeluted with hexanes (100 mL) followed by a mixture of hexanes/ethylacetate (95:5, 100 mL). The hexane/ethyl acetate fractions were combinedand concentrated under vacuum to give a gummy mass.

The product POH carbamate exhibited a weight of 120 mg and a yield of31%. ¹H-NMR (400 MHz, CDCl₃): δ 0.9 (m, 2H), 1.4 (m, 2H), 1.7 (m, 7H*),1.95 (m, 8H*), 2.1 (m, 4H), 2.3 (s, 3H), 4.4 (d, 2H), 4.7 (dd, 2H), 5.6(br d, 2H), 6.6 (s, 1H), 7.0 (br s, 1H), 7.12 (d, 1H), 7.19 (d, 1H), 7.4(d, 2H), 7.85 (d, 2H); MS, m/e: 751.8 (M⁺ 3%), 574.3 (100%), 530.5(45%), 396 (6%). * N.B. further 2H overlapping from presumed impuritydiscounted in NMR integration.

Example 2 In Vitro Cytotoxicity Studies of Dimethyl Celecoxib bisPOHCarbamate (POH-DMC)

First cytotoxicity assays were carried out after cells were treated withdimethyl-celecoxib (DMC) alone. FIG. 1 shows the results of the MTTcytotoxicity assays performed on human malignant glioma cells U87, A172and U251 with DMC alone.

Then U87, A172 and U251 cells were treated with dimethyl celecoxibbisPOH carbamate (POH-DMC) (e.g., synthesized by the method in Example1), and the MTT cytotoxicity assays performed (FIG. 2). The resultssuggest that POH carbamate POH-DMC exhibited much better cytotoxicitythan DMC alone.

Example 3 Synthesis of Temozolomide POH Carbamate (3-methyl4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carbonyl)-carbamicacid-4-isopropenyl cyclohex-1-enylmethyl ester)

The reaction scheme is the following:

Oxalyl chloride (0.13 grams, 1.0 mmol) was added slowly to a mixture oftemozolomide (OChem Incorporation, 0.1 grams, 0.5 mmol) in1,2-dichloroethane (10 mL) over a period of 2 minutes while maintainingthe temperature at 10° C. under N₂. The reaction mixture was allowed towarm to room temperature and then heated to reflux for 3 hours. Theexcess of oxalyl chloride and 1,2-dichloroethane were removed byconcentration under vacuum. The resulting residue was re-dissolved in1,2-dichlorethane (15 mL) and the reaction mixture was cooled to 10° C.under N₂. A solution of perillyl alcohol (0.086 grams, 0.56 mmol) in1,2-dichloroethane (3 mL) was added over a period of 5 minutes. Thereaction mixture was allowed to warm to room temperature and stirred for14 hours. 1,2-dichloroethane was concentrated under vacuum to give aresidue, which was triturated with hexanes. The resulting yellow solidwas filtered and washed with hexanes. Weight: 170 mg; Yield: 89%. ¹H-NMR(400 MHz, CDCl₃): δ 1.4-2.2 (m, 10H), 4.06 (s, 3H), 4.6-4.8 (m, 4H),5.88 (br s, 1H), 8.42 (s, 1H), 9.31 (br s, 1H); MS, no molecular ionpeak was observed. m/e: 314 (100%), 286.5 (17%), 136 (12%).

Alternatively, temozolomide POH carbamate was synthesized according tothe following procedure. Oxalyl chloride (0.13 grams, 1.0 mmol) wasadded slowly to a mixture of temozolomide (OChem Incorporation, 0.1grams, 0.5 mmol) in 1,2-dichloroethane (10 mL) over a period of 2minutes while maintaining the temperature at 10° C. under N₂. Thereaction mixture was allowed to warm to room temperature and then heatedto reflux for 3 hours. The excess of oxalyl chloride and1,2-dichloroethane were removed by concentration under vacuum. Theresulting residue was re-dissolved in 1,2-dichlorethane (15 mL) and thereaction mixture was cooled to 10° C. under N₂. A solution of perillylalcohol (0.086 grams, 0.56 mmol) in 1,2-dichloroethane (3 mL) was addedover a period of 5 minutes. The reaction mixture was allowed to warm toroom temperature and stirred for 14 hours. 1,2-Dichloroethane wasconcentrated under vacuum to give a residue, which was purified by ashort silica-plug column (column dimensions: diameter: 2 cm, height: 3cm, silica: 230-400 mesh) and eluted with a mixture of hexanes/ethylacetate (1:1, 100 mL). The hexane/ethyl acetate fractions were combinedand concentrated under vacuum to give a white solid residue which wastriturated with heptanes and filtered to obtain a white solid. Weight:170 mg; Yield: 89%. ¹H-NMR (400 MHz, CDCl₃): 1.4-2.2 (m, 10H), 4.06 (s,3H), 4.6-4.8 (m, 4H), 5.88 (br s, 1H), 8.42 (s, 1H), 9.31 (br s, 1H);MS, no molecular ion peak was observed, m/e: 314 (100%), 286.5 (17%),136 (12%).

Example 4 In Vitro Cytotoxicity Studies of Temozolomide POH Carbamate(POH-TMZ)

First cytotoxicity assays were carried out after cells were treated withtemozolomide (TMZ) alone, the standard alkylating agent used in thetreatment of malignant gliomas. FIG. 3 shows the results of the MTTcytotoxicity assays performed on human malignant glioma cells U87, A172and U251 with TMZ alone. Increasing concentrations of TMZ had minimalcytotoxicity towards the cell lines tested.

Then TMZ-resistant glioma cell lines U87, A172 and U251 cells weretreated with temozolomide POH carbamate (POH-TMZ) (e.g., synthesized bythe method in Example 3). The MTT assay results (FIG. 4) showed that POHcarbamate POH-TMZ exhibited substantially higher kill rates of thevarious human glioma cells compared to TMZ alone.

Example 5 Synthesis of Rolipram POH Carbamate(4-(3-cyclopentyloxy-4-methoxy phenyl)-2-oxo-pyrrolidine-1-carboxylicacid 4-isopropenyl cyclohex-1-enylmethyl ester)

The reaction scheme is the following:

Phosgene (20% in toluene, 13 ml, 26.2 mmol) was added to a mixture ofperillyl alcohol (2.0 grams, 13.1 mmol) and potassium carbonate (5.4grams, 39.1 mmol) in dry toluene (30 mL) over a period of 30 minuteswhile maintaining the temperature between 10° C. to 15° C. The reactionmixture was allowed to warm to room temperature and stirred for 8.0hours under N₂. The reaction mixture was quenched with water (30 mL) andthe organic layer separated. The aqueous layer was extracted withtoluene (20 mL) and the combined organic layer washed with water (50mL×2), brine (15%, 30 mL) and dried over sodium sulfate (20 grams). Thefiltered organic layer was concentrated under vacuum to give perillylchloroformate as an oil. Weight: 2.5 grams; Yield: 89%. ¹H-NMR (400 MHz,CDCl₃): δ 1.5 (m, 1H), 1.7 (s, 3H), 1.8 (m, 1H), 2.0 (m, 1H), 2.2 (m,4H), 4.7 (dd, 4H); 5.87 (m, 1H).

Butyl lithium (2.5 M, 0.18 mL, 0.45 mmol) was added to a solution ofrolipram (GL synthesis, Inc., 0.1 grams, 0.36 mmol) in dry THF at −72°C. over a period of 5 minutes under N₂. After the reaction mixture wasstirred for 1.0 hours at −72° C., perillyl chloroformate (dissolved in 4mL THF) was added over a period of 15 minutes while maintaining thetemperature at −72° C. The reaction mixture was stirred for 2.5 hoursand quenched with saturated ammonium chloride (5 mL). The reactionmixture was allowed to warm to room temperature and extracted with ethylacetate (2×15 mL). The combined organic layer was washed with water (15mL), brine (15%, 15 mL), and then dried over sodium sulfate. Thefiltered organic layer was concentrated to give an oil which waspurified by column chromatography [column dimensions: diameter: 1.5 cm,height: 10 cm, silica: 230-400 mesh] and eluted with a mixture of 8%ethyl acetate/hexanes (100 mL) followed by 12% ethyl acetate/hexanes(100 mL). The 12% ethyl acetate/hexanes fractions were combined andconcentrated under vacuum to yield a gummy solid. Weight: 142 mg; Yield:86%. ¹H-NMR (400 MHz, CDCl₃): δ 1.5 (m, 1H), 1.6 (m, 2H), 1.7 (s, 3H),1.9 (m, 6H), 2.2 (m, 5H), 2.7 (m, 1H), 2.9 (m, 1H), 3.5 (m, 1H), 3.7 (m,1H), 3.8 (s, 3H), 4.2 (m, 1H), 4.7 (m, 6H), 5.8 (br s, 1H), 6.8 (m, 3H);MS, m/e: 452.1 (M⁺¹ 53%), 274.1 (100%), 206.0 (55%).

Example 6 In Vitro Cytotoxicity Studies of Rolipram POH Carbamate(POH-Rolipram)

To compare the cytotoxicity of Rolipram POH Carbamate (POH-Rolipram)(e.g., synthesized by the method in Example 5) with rolipram, a type IVphosphodiesterase inducing differentiation and apoptosis in gliomacells, A172, U87, U251 and LN229 human glioma cells were treated witheither POH-Rolipram or rolipram for 48 hours. The MTT assay results areshown in FIGS. 5 to 8. POH-Rolipram exhibited substantially higher killrates compared to rolipram alone for each of the several different humanglioma cell types. FIG. 5 shows the MTT assay for increasingconcentrations of rolipram and POH-rolipram for A-172 cells. Rolipramalone demonstrates an IC50 of approximately 1000 uM (1 mM). In thepresence of POH-rolipram, IC50 is achieved at concentrations as low as50 uM. FIG. 6 shows the MTT assay for increasing concentrations ofrolipram with U-87 cells. IC50 is not met at 1000 uM. On the other hand,IC50 is achieved at 180 uM with POH-rolipram. FIG. 7 shows that IC50 forrolipram alone for U251 cells is achieved at 170 uM; plateaucytotoxicity is reached at 60%. POH-rolipram achieves IC50 at 50 uM,with almost 100% cytoxicity at 100 uM. FIG. 8 shows that IC50 forrolipram alone for LN229 cells is not achieved even at 100 uM. On theother hand, IC50 for POH-rolipram is achieved at 100 uM, with almost100% cytotoxicity at 10 uM.

Example 7 In Vivo Tumor Growth Inhibition by POH Fatty Acid Derivatives

Inhibition of tumor growth by butyryl-POH was studied in a nude mousesubcutaneous glioma model. Mice were injected with U-87 glioma cells(500,000 cells/injection) and allowed to form a palpable nodule over twoweeks. Once palpable nodule was formed, the mice were treated with localapplication of various compounds as indicated in FIGS. 9A and 9B via aQ-tip (1 cc/application/day) over a period of 8 weeks. FIG. 9A shows theimages of subcutaneous U-87 gliomas in nude mice treated withbutyryl-POH, purified (S)-perillyl alcohol having a purity greater than98.5% (“purified POH”), POH purchased from Sigma chemicals, or phosphatebuffered saline (PBS; negative control). FIG. 9B shows average tumorgrowth over time (total time period of 60 days). Butyryl-POHdemonstrated the greatest inhibition of tumor growth, followed bypurified POH and Sigma POH.

Example 8 In Vitro Cytotoxicity Studies of Temozolomide (TMZ) andTemozolomide POH Carbamate (POH-TMZ) on TMZ Sensitive and ResistantGlioma Cells

Colony forming assays were carried out after cells were treated with TMZalone, POH alone, and the TMZ-POH conjugate. The colony forming assayswere carried out as described in Chen T C, et al. Green teaepigallocatechin gallate enhances therapeutic efficacy of temozolomidein orthotopic mouse glioblastoma models. Cancer Lett. 2011 Mar. 28;302(2):100-8. FIG. 10 shows the results of the colony forming assaysperformed on TMZ sensitive (U251) and TMZ resistant (U251TR) U251 cellswith TMZ or TMZ-POH. TMZ demonstrated cytotoxicity towards TMZ sensitiveU251 cells, but had minimal cytotoxicity towards TMZ resistant U251cells. TMZ-POH demonstrated cytotoxicity towards both TMZ sensitive andTMZ resistant U251 cells.

FIG. 11 shows the results of the colony forming assays performed on TMZsensitive (U251) and TMZ resistant (U251TR) U251 cells with POH. POHdemonstrated cytotoxicity towards both TMZ sensitive and TMZ resistantU251 cells. POH-TMZ (FIG. 10) exhibited substantially greater potencycompared to POH alone (FIG. 11) in the colony forming assays.

Example 9 In Vitro Cytotoxicity Studies of Temozolomide POH Carbamate(POH-TMZ) on U251 Cells, U251TR Cells, and Normal Astrocytes

MTT cytotoxicity assays were carried out after cells were treated withthe TMZ-POH conjugate. The MTT cytotoxicity assays were carried out asdescribed in Chen T C, et al. Green tea epigallocatechin gallateenhances therapeutic efficacy of temozolomide in orthotopic mouseglioblastoma models. Cancer Lett. 2011 Mar. 28; 302(2):100-8. FIG. 12shows the results of the MTT cytotoxicity assays performed on TMZsensitive cells (U251), TMZ resistant cells (U251TR) and normalastrocytes. TMZ-POH demonstrated cytotoxicity towards both TMZ sensitiveand TMZ resistant U251 cells, but not towards normal astrocytes.

Example 10 In Vitro Cytotoxicity Studies of Temozolomide POH Carbamate(POH-TMZ) on BEC, TuBEC, and Normal Astrocytes

MTT cytotoxicity assays were carried out after cells were treated withthe TMZ-POH conjugate. The MTT cytotoxicity assays were carried out asdescribed in Chen T C, et al. Green tea epigallocatechin gallateenhances therapeutic efficacy of temozolomide in orthotopic mouseglioblastoma models. Cancer Lett. 2011 Mar. 28; 302(2):100-8. FIG. 13shows the results of the MTT cytotoxicity assays performed on normalastrocytes, brain endothelial cells (BEC; confluent and subconfluent),and tumor brain endothelial cells (TuBEC). TMZ-POH did not inducesignificant cytotoxicity on normal astrocytes, confluent BEC, or TuBEC.Mild to moderate cytotoxicity was demonstrated in subconfluent BEC athigh concentrations of TMZ-POH.

Example 11 In Vitro Cytotoxicity Studies of Temozolomide (TMZ) andTemozolomide POH Carbamate (POH-TMZ) on USC-04 Glioma Cancer Stem Cells

MTT cytotoxicity assays were carried out after cells were treated withthe TMZ alone, POH alone, or the TMZ-POH conjugate. The MTT cytotoxicityassays were carried out as described in Chen T C, et al. Green teaepigallocatechin gallate enhances therapeutic efficacy of temozolomidein orthotopic mouse glioblastoma models. Cancer Lett. 2011 Mar. 28;302(2):100-8. FIG. 14 shows the results of the MTT cytotoxicity assaysperformed on USC-04 glioma cancer stem cells. TMZ did not inducesignificant cytotoxicity with increasing concentrations (0-400 uM).TMZ-POH demonstrated evidence of cytotoxicity with IC50 at 150 uM. FIG.15 shows the results of the MTT cytotoxicity assays performed on USC-04glioma cancer stem cells treated with POH. POH demonstrated cytotoxicityon USC-04 with increasing concentrations (0-2 mM).

Example 12 In Vitro Cytotoxicity Studies of Temozolomide (TMZ) andTemozolomide POH Carbamate (POH-TMZ) on USC-02 Glioma Cancer Stem Cells

MTT cytotoxicity assays were carried out after cells were treated withthe TMZ alone, POH alone, or the TMZ-POH conjugate. The MTT cytotoxicityassays were carried out as described in Chen T C, et al. Green teaepigallocatechin gallate enhances therapeutic efficacy of temozolomidein orthotopic mouse glioblastoma models. Cancer Lett. 2011 Mar. 28;302(2):100-8. FIG. 16 shows the results of the MTT cytotoxicity assaysperformed on USC-02 glioma cancer stem cells. TMZ did not inducesignificant cytotoxicity with increasing concentrations (0-400 uM).TMZ-POH demonstrated evidence of cytotoxicity with IC50 at 60 uM. FIG.17 shows the results of the MTT cytotoxicity assays performed on USC-02glioma cancer stem cells treated with POH. POH demonstrated cytotoxicityon USC-02 with increasing concentrations (0-2 mM).

Example 13 In Vitro Studies of ER Stress by Temozolomide POH Carbamate(POH-TMZ) on TMZ Sensitive and Resistant Glioma Cells

Western blots were performed after TMZ sensitive and resistant gliomacells were treated with the TMZ-POH conjugate for 18 hr. FIG. 18 shows awestern blot demonstrating that TMZ-POH induces ER stress (ERS) in TMZsensitive and resistant U251 glioma cells. Activation of the proapopticprotein CHOP was shown at concentrations as low as 60 uM of TMZ-POH.

The scope of the present invention is not limited by what has beenspecifically shown and described hereinabove. Those skilled in the artwill recognize that there are suitable alternatives to the depictedexamples of materials, configurations, constructions and dimensions.Numerous references, including patents and various publications, arecited and discussed in the description of this invention. The citationand discussion of such references is provided merely to clarify thedescription of the present invention and is not an admission that anyreference is prior art to the invention described herein. All referencescited and discussed in this specification are incorporated herein byreference in their entirety. Variations, modifications and otherimplementations of what is described herein will occur to those ofordinary skill in the art without departing from the spirit and scope ofthe invention. While certain embodiments of the present invention havebeen shown and described, it will be obvious to those skilled in the artthat changes and modifications may be made without departing from thespirit and scope of the invention. The matter set forth in the foregoingdescription and accompanying drawings is offered by way of illustrationonly and not as a limitation.

What is claimed is:
 1. A pharmaceutical composition comprising aperillyl alcohol carbamate.
 2. The pharmaceutical composition of claim1, wherein the perillyl alcohol carbamate is perillyl alcohol conjugatedwith a therapeutic agent.
 3. The pharmaceutical composition of claim 2,wherein the therapeutic agent is a chemotherapeutic agent.
 4. Thepharmaceutical composition of claim 3, wherein the chemotherapeuticagent is selected from the group consisting of a DNA alkylating agent, atopoisomerase inhibitor, an endoplasmic reticulum stress inducing agent,a platinum compound, an antimetabolite, an enzyme inhibitor, and areceptor antagonist.
 5. The pharmaceutical composition of claim 2,wherein the therapeutic agent is selected from the group consisting ofdimethyl celocoxib (DMC), temozolomide (TMZ) and rolipram.
 6. Thepharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition is administered before, during or after radiation.
 7. Thepharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition is administered before, during or after the administrationof a chemotherapeutic agent.
 8. The pharmaceutical composition of claim1, wherein the pharmaceutical composition is administered by inhalation,intranasally, orally, intravenously, subcutaneously or intramuscularly.9. The pharmaceutical composition of claim 1, wherein the perillylalcohol carbamate is 4-(Bis-N,N′-4-isopropenyl cyclohex-1-enylmethyloxycarbonyl[5-(2,5-dimethylphenyl)-3-trifluoromethylpyrazol-1-yl]benzenesulfonamide.
 10. Thepharmaceutical composition of claim 1, wherein the perillyl alcoholcarbamate is 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-oxo-pyrrolidine-1-carboxylic acid 4-isopropenylcyclohex-1-enylmethyl ester.
 11. The pharmaceutical composition of claim1, wherein the perillyl alcohol carbamate is 3-methyl4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carbonyl)-carbamicacid-4-isopropenyl cyclohex-1-enylmethyl ester.
 12. A method fortreating a disease in a mammal, comprising the step of delivering to themammal a therapeutically effective amount of a perillyl alcoholcarbamate.
 13. The method of claim 12, wherein the disease is cancer.14. The method of claim 13, wherein the cancer is a tumor of the nervoussystem.
 15. The method of claim 14, wherein the tumor is a glioblastoma.16. The method of claim 12, further comprising the step of treating themammal with radiation.
 17. The method of claim 12, further comprisingthe step of delivering to the mammal a chemotherapeutic agent.
 18. Themethod of claim 12, wherein the perillyl alcohol carbamate isadministered by inhalation, intranasally, orally, intravenously,subcutaneously or intramuscularly.
 19. The method of claim 12, whereinthe perillyl alcohol carbamate is perillyl alcohol conjugated with atherapeutic agent.
 20. The method of claim 19, wherein the therapeuticagent is a chemotherapeutic agent.
 21. The method of claim 20, whereinthe chemotherapeutic agent is selected from the group consisting of aDNA alkylating agent, a topoisomerase inhibitor, an endoplasmicreticulum stress inducing agent, a platinum compound, an antimetabolite,an enzyme inhibitor, and a receptor antagonist.
 22. The method of claim19, wherein the therapeutic agent is selected from the group consistingof dimethyl celocoxib (DMC), temozolomide (TMZ) and rolipram.
 23. Themethod of claim 12, wherein the perillyl alcohol carbamate is selectedfrom the group consisting of (a) 4-(bis-N,N′-4-isopropenylcyclohex-1-enylmethyloxy carbonyl[5-(2,5-dimethylphenyl)-3-trifluoromethylpyrazol-1-yl]benzenesulfonamide; (b)4-(3-cyclopentyloxy-4-methoxy phenyl)-2-oxo-pyrrolidine-1-carboxylicacid 4-isopropenyl cyclohex-1-enylmethyl ester; and (c) 3-methyl4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carbonyl)-carbamicacid-4-isopropenyl cyclohex-1-enylmethyl ester.
 24. A process for makinga POH carbamate, comprising the step of reacting a first reactant ofperillyl chloroformate with a second reactant selected from the groupconsisting of dimethyl celocoxib (DMC), temozolomide (TMZ) and rolipram.25. The process of claim 24, wherein the second reactant is dimethylcelocoxib.
 26. The process of claim 25, wherein the reaction is carriedout in the presence of acetone and a catalyst of potassium carbonate.27. The process of claim 24, wherein the second reactant is rolipram.28. The process of claim 27, wherein the reaction is carried out in thepresence of tetrahydrofuran and a catalyst of n-butyl lithium.
 29. Theprocess of claim 24, wherein the perillyl chloroformate is prepared byreacting perillyl alcohol with phosgene.